This work is designed to understand how proteins encoded by two deafness genes-cadherin 23 and protocadherin 15-assemble to form the mechanosensory apparatus of hair cells in the auditory and vestibular systems. Each hair cell has a bundle of actin-based stereocilia arranged with increasing heights;each stereocilium of a cell extends a filamentous 'tip link'to the next taller stereocilium. Movement of the bundle tightens tip links;they in turn pull open force-gated ion channels that open to depolarize the cell. Thus tip links are a key component at the heart of inner ear function-to turn sound and head movement into neural signals. Recent evidence indicates that each tip link is composed of cadherin 23 and protocadherin 15 arranged in an antiparallel hetero-tetrameric filament. While the homomeric binding of classical cadherins is understood, these tip-link cadherins lack the key amino acids that mediate such binding. Moreover, mutations in either protein cause Usher Syndrome, characterized by congenital deafness and progressive blindness, but it is not known how these mutations cause hearing loss. We will investigate the binding between these cadherins, by solving the crystal structures of the distal ends individually and then as a heterotetrameric complex. We have already solved four structures for the cadherin 23 N-terminus: for wild-type and mutant forms of the proteins, and in high and low Ca2+ concentration. We will extend this to the protocadherin 15 N terminus, to understand how both Ca2+ and mutations affect binding and tip-link integrity. With crystal structures in hand, we will use steered molecular dynamics calculations to understand how these cadherins unfold in response to high tension, and how Ca2+ concentration and deafness-causing mutations affect the unfolding force. Initial work shows that removing Ca2+ or mutating Ca2+-binding residues allows cadherins to unfold at lower force, making them more susceptible to loud noise. We will also use molecular dynamics to explore the predicted heteromeric binding interface by asking what forces are needed to pull apart the tip link and how Ca2+ maintains that bond. The molecular structure of the bond between cadherin 23 and protocadherin 15 suggested by these studies will be tested by mutagenesis experiments, to see which amino acids are critical for binding in vitro and which are required to prevent regeneration of tip links by capping the free ends. PUBLIC HEALTH RELEVANCE: Usher's Syndrome, a devastating inherited disorder characterized by congenital deafness and progressive blindness, is caused by mutations in genes encoding the proteins cadherin 23 and protocadherin 15. This project is directed at understanding what these cadherins do in the inner ear;specifically, which forms are part of the sensory apparatus of cochlear receptor cells, and how they assemble to create the apparatus. The results will help us design strategies for treating inherited deafness.